The possibility of bio-terrorism, outbreak of Severe Acute Respiratory Syndrome (SARS) and Bird Flu virus, continuing spread of HIV/AIDS, and the emergence of pathogenic strains resistant against current medications compel investigators to look for new protective measures including biodefense strategies against these threats. Immune activation is an effective as well as protective approach for biodefense strategies for emerging infectious diseases (Hackett, C. J. J. Allergy and Clin. Immunol., 2003, 112:686-694). Although the human immune system possesses a wide array of microbial detection and host defense mechanisms, pathogen evasion of the immune surveillance and destruction system is the norm. The situation is not different with cancer, as most of the tumor cells are not detected and escape immune surveillance (Wajchman, H. J. et al. Cancer Res., 2004, 64:1171-1180).
It is the innate immune system components that detect the pathogens or the non-self intruders, with the help of the specific receptors, and respond immediately by activation of the immune competent cells, synthesis of cytokines and chemokines, and release of inflammatory mediators to eliminate or contain the intruders. Innate immune activation also triggers and paves the way for the adaptive immune response by antigen-specific T and B lymphocytes. The natural killer (NK) cells play a decisive role in the innate immune defense against virus infected and malignant cells by virtue of their ability to recognize and destroy abnormal cells, pending the development of adaptive immunity (Smith, H. R. et al. Proc. Natl. Acad. Sci. USA, 2002, 99:8826-8831; Moser, J. M. et al. Curr. Opin. Immunol., 2002, 14:509-516).
Several compounds activate the immune system, such as microbial lipopolysaccharides (LPS), double-stranded RNA and DNA oligonucleotides containing unmethylated CpG motifs have been reported earlier (Hackett, C. J. J. Allergy and Clin. Immunol., 2003, 112:686-694; Kandimalla, E. R. et al. Proc. Natl. Acad. Sci. USA, 2003, 100:14303-14308). Stimulation of multiple receptors is reported to exhibit synergistic effect in cytokine production (Gao, J. J. et al. J. Immunol., 2001, 166:6855-6860). The complement activation cascade is another integral part of the innate immune system in which the cellular pathogens, such as intracellular bacteria, are coated with complement components (opsonization) and readily undergo phagocytosis. Thus, complement activation plays an important role in microbial killing and is essential for transport and clearance of immune complexes.
The type of adaptive immune response (also known as the specific or acquired immune response) that is generated to infection or other antigenic challenge can generally be distinguished by the subset of T helper (Th) cells involved in the response. The Th1 response (the cellular or cell-mediated response) is responsible for classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs), whereas the Th2 response (the humoral response) functions more effectively as a helper for B-cell activation. The type of immune response to an antigen is generally determined by the cytokines produced by the cells responding to the antigen. Differences in the cytokines secreted by Th1 and Th2 cells are believed to reflect different biological functions of these two subsets.
It has become clear that cytokines play crucial roles in regulating various aspects of immune responses. Among the cytokines, interleukin (IL)-12 plays a central role in coordinating innate and cell-mediated adaptive immunity (Watford, W. T. et al. Cytokine Growth Factor Rev., 2003, 14:361-368). Prophylactic as well as post-exposure protection by immune stimulation have been demonstrated (Walker, P. S. et al. Proc. Natl. Acad. Sci. USA, 1999, 96:6970-6975; Juffermans, N. P. et al Infect. Immunol., 2002, 70:147-152). Usually, these protective measures are correlated with synthesis of IL-12 and interferon (IFN)-γ, the cytokines of the Th1 pathway of T cell differentiation associated with the adaptive immune system (Gramzinski, A. M. et al. Infect. Immunol., 2001, 69:1643-1649).
The Th1 subset may be particularly suited to respond to viral infections and intracellular pathogens because it secretes IL-2 and IFN-gamma, which activate CTLs. The Th2 subset may be more suited to respond to free-living bacteria and helminthic parasites and may mediate allergic reactions, since IL-4 and IL-5 are known to induce IgE production and eosinophil activation, respectively. In general, Th1 and Th2 cells secrete distinct patterns of cytokines and, therefore, one type of response can moderate the activity of the other type of response. A shift in the Th1/Th2 balance can result in an allergic response, for example, or, alternatively, in an increased CTL response.
Immunization of a host animal against a particular antigen has been accomplished traditionally by repeatedly vaccinating the host with an immunogenic form of the antigen. While most current vaccines elicit effective humoral (antibody, or “Th2-type”) responses, they fail to elicit cellular responses (in particular, major histocompatibility complex (MHC) class I-restricted CTL, or “Th1-type” responses) which are generally absent or weak. For many infectious diseases, such as tuberculosis and malaria, Th2-type responses are of little protective value against infection. Moreover, antibody responses are inappropriate in certain indications, most notably in allergy where an antibody response can result in anaphylactic shock. Proposed vaccines using small peptides derived from the target antigen and other currently used antigenic agents that avoid use of potentially infective intact viral particles, do not always elicit the immune response necessary to achieve a therapeutic effect. The lack of a therapeutically effective human immunodeficiency virus (HIV) vaccine is an unfortunate example of this failure.
Innate immunity mediated by macrophages, neutrophils and natural killer (NK) cells is the first line of host defense mechanism against microbial invasion. The innate immune system targets the structurally conserved pathogen-associated molecular patterns (PAMPs) through specific germ-line encoded receptors called pattern recognition receptors (PRRs) (Aderem, A. and Ulevitch, R. J. Nature, 2000, 406:782-787). Augmentation of the immune system with natural as well as synthetic immune stimulators offers a distinct advantage over conventional therapies especially with weakened immune system and antibiotic resistance.
Several preclinical and clinical investigations have indicated the usefulness of β-glucans, a class of biological response modifiers (BRMs), for acceleration of wound healing and against orchestration of the systemic inflammatory response syndrome and septic shock (Ross, G. D. et al. Immunopharmacology, 1999, 42:61-74; Williams, D. L. Mediators Inflamm., 1997, 6:247-250; Hetland, G. Curr. Med. Chem., 2003, 2:135-146). β-glucans are potent stimulators of innate immune system in invertebrates, while in mammals they are potent activators of the complement system. These polymers have therapeutic potential because of their effects on the immune system that may include anti-tumor and anti-infective activities as well as protection against fungal, bacterial, viral, and protozoan infections. Soluble and particulate β-glucans interact with cognate receptors on macrophages stimulating the syntheses of cytokines, chemokines and reactive oxygen intermediates (Ganter, B. N. et al. J. Expt. Med., 2003, 197:1107-1117). The major receptors reported for β-glucan recognition/binding on macrophages are complement receptor 3 (CD11b/CD18 or CR3), Dectin-1 and Toll-like receptors (TLRs) 2 and 6. Although lactosylceramide and scavenger receptors are also identified in β-glucan recognition, their function is not well-documented (Willment, J. A. et al. J. Biol. Chem., 2001, 276:43813-43823).
Toll like receptors (TLRs) are part of the large super family of Toll-Interleukin (IL)-1 receptors (TIRs) possessing the cytoplasmic motif for the intracellular signaling function. These molecules provide a first line host defense and have been implicated in infectious and autoimmune diseases in a variety of organisms ranging from flies to mammals. It is now accepted that TLRs are the principal signaling molecules through which mammals sense infection (Beutler, B. et al. J Leukocyte Biol., 2003, 74:479-485). In mammals, 12 different TLRs have been identified each recognizing distinct PAMPs (Akira, S. and Sato, S. Scand. J. Infect. Dis., 2003, 35:555-562). All TLRs, IL-1 receptor and other TIR domain containing receptors, with the exception of TLR3, share a common signaling pathway that depends on the adaptor myeloid differentiation factor 88 (MyD88) (Ganter, B. N. et al. J. Expt. Med., 2003, 197:1107-1117; Mukhopadhyay, S. et al. Immunol., 2004, 112:521-530). Besides MyD88, several adaptor molecules have recently been reported and the differential utilization of these adaptor molecules may provide the specificity for the TLR signaling (Akira, S. J. Biol. Chem., 2003, 278:38105-38108). Evidence for the physical and/or functional interactions among TLRs, and between TLR and other surface receptors, has become available. Gantner et al. have reported collaborative induction of dectin-1 and TLR by β-glucan stimulation as well as the synergistic interaction between these two receptors on NF-κB activation (Ganter, B. N. et al. J. Expt. Med., 2003, 197:1107-1117).
TLR mediated cytokine production depends on its down stream mediators such as IL-1R-associated kinase (IRAK)-4 and TNF receptor-associated factor-6 (TRAF-6) that activate JNK and nuclear factor (NF)-κB (Akira, S. J. Biol. Chem., 2003, 278:38105-38108). NF-κB is a ubiquitous transcription factor that regulates the cytokine gene expression in many immune effecter cells. In most cells, NF-κB is usually present in cytoplasm as latent, inactive and bound to the inhibitory protein κB (I-κB) (Ghosh, S, and Karin, M., Cell, 2002, 109:S81-96). It is activated by a variety of stimuli such as pro-inflammatory cytokines, viral products, lipopolysaccharides, plant derived compounds such as taxol, as well as pathogen and non-pathogen derived β-glucans (Akira, S. and Sato, S. Scand. J. Infect. Dis., 2003, 35:555-562; Young, S. H. et al. J. Biol. Chem., 2001, 276:20781-20787; Lebron, F. et al. J Biol Chem., 2003, 278:25001-25008). On stimulation, I-κBα is phosphorylated and rapidly degraded through proteasomal mechanisms which in turn release the active NF-κB so as to translocate to the nucleus and bind to DNA to initiate cytokine/chemokine gene transcription (Ghosh, S, and Karin, M., Cell, 2002, 109:S81-96; Auphan, N. et al. Science, 1995, 270:232-233).
Immunostimulating properties of glucans have been ascribed to be due to the β-glycosidic linkages, degree of branching and solution conformation (Mueller, A et al. Glycobiology, 2000, 10:339-346). The present inventors have characterized and reported the immunostimulating properties of a (1,4)-α-D-glucan, RR1, a novel polysaccharide obtainable from the medicinal plant Tinospora cordifolia (Nair, P. K. et al. Int. Immunopharmacol., 2004, 4:1645-1659). This novel α-glucan is water soluble and has (1,4)-α-D-glycosidic linkages in the main chain with a (1,6)-α-D-glycosidic linked side chains at an interval of 6 to 7 glucose units. It is non-cytotoxic to normal cells as well as tumor cell lines (CEM, CEM/VLB) even up to 1000 μg/ml and activates the human lymphocyte subsets at varying levels. The activation of NK cells, one of the major arms of innate immunity, was demonstrated by the increased level of killing of target cells by the RR1-treated lymphocytes in a functional assay. The cytokine profile upon RR1 stimulation demonstrates the much desired Th1 pathway of T helper cell differentiation along with high level of induction of regulatory cytokines which may be a self control mechanism of the over production of Th1 response. Its water solubility, non-cytotoxic nature, and its herbal origin indicate the clinical potential of RR1 for immune stimulation.